THE EXPRESSIONS OF HBV X GENE AND ets-2, IGF-Ⅰ, c-myc AND N-ras ONCOGENES IN HUMAN HEPATOCELLULAR CARCINOMA AND TUMOR-ADJACENT TISSUES

The expressions of HBV X gene and ets-2, IGF-I, c-myc and N-ras were studied in 7 pairs of human primary hepatocellular carcinoma (PHC) and tumor-adjacent tissues, using RNA hybridization and im-munoblot methods. The results showed that specific 17 and 28 kD HBV X gene products (HBxAg) were existed in a portion of PHC and tumor-adjacent tissues. The 17 kD HBxAg was detected in the sera of 3 patients who also had 17 kD HBxAg in their liver tissues. Multiple expressions of oncogenes such as ets-2, c-myc and N-ras were observed in PHC and tumor-adjacent tissues that had HBxAg expressed, indicating HBxAg might function as a transactivator in the course of intracellular proto-oncogene activation. It is also observed that in some tumor-adjacnet tissues the expressions of ets-2, c-myc and N-ras were higher than those in corresponding PHC. The relationship of HBxAg to the expression of est-2, IGF-Ⅱ, c-myc and their possible roles in the carcinogenesis of PHC are discussed.

Targeting c-Myc as a novel approach for hepatocellular carcinoma

Hepatocelluar carcinoma(HCC) is the most lethal cancer in the world.Most HCC over-express c-Myc,which plays a critical role in regulating cellular growth,differentiation and apoptosis in both normal and neoplastic cells. c-Myc is among the most frequently overexpressed genes in human cancers.Overexpression of c-Myc in hepatic cells leads to development of hepatocellular carcinoma.Here,we review the current progress in understanding physiologic function and regulation of c-Myc as well as its role in hepatic carcinogenesis and discuss the association of c-Myc activation in chronic hepatitis B infection and the upregulation of HIF-1/VEGF.We also explore the possibility of treating HCC by inhibiting c-Myc and examine the pros and cons of such an approach.Although this strategy is currently not available in clinics,with recent advances in better drug design,pharmacokinetics and pharmacogenetics,inhibition of c-Myc might become a novel therapy for HCC in the future.

Reactive oxygen species-induced activation of Yes-associated protein-1 through the c-Myc pathway is a therapeutic target in hepatocellular carcinoma

BACKGROUND The Hippo signaling pathway regulates organ size by regulating cell proliferation and apoptosis with terminal effectors including Yes-associated protein-1(YAP-1).Dysregulation in Hippo pathway has been proposed as one of the therapeutic targets in hepatocarcinogenesis.The levels of reactive oxygen species(ROS)increase during the progression from early to advanced hepatocellular carcinoma(HCC).AIM To study the activation of YAP-1 by ROS-induced damage in HCC and the involved signaling pathway.METHODS The expression of YAP-1 in HCC cells(Huh-7,HepG2,and SNU-761)was quantified using real-time polymerase chain reaction and immunoblotting.Human HCC cells were treated with H2O2,which is a major component of ROS in living organisms,and with either YAP-1 small interfering RNA(siRNA)or control siRNA.To investigate the role of YAP-1 in HCC cells under oxidative stress,MTS assays were performed.Immunoblotting was performed to evaluate the signaling pathway responsible for the activation of YAP-1.Eighty-eight surgically resected frozen HCC tissue samples and 88 nontumor liver tissue samples were used for gene expression analyses.RESULTS H2O2 treatment increased the mRNA and protein expression of YAP-1 in HCC cells(Huh-7,HepG2,and SNU-761).Suppression of YAP-1 using siRNA transfection resulted in a significant decrease in tumor proliferation during H2O2 treatment both in vitro and in vivo(both P<0.05).The oncogenic action of YAP-1 occurred via the activation of the c-Myc pathway,leading to the upregulation of components of the unfolded protein response(UPR),including 78-kDa glucoseregulated protein and activating transcription factor-6(ATF-6).The YAP-1 mRNA levels in human HCC tissues were upregulated by 2.6-fold compared with those in nontumor tissues(P<0.05)and were positively correlated with the ATF-6 Levels(Pearson’s coefficient=0.299;P<0.05).CONCLUSION This study shows a novel connection between YAP-1 and the UPR through the c-Myc pathway during oxidative stress in HCC.The ROS-induced activation of YAP-1 via the c-Myc pathway,which leads to the activation of the UPR pathway,might be a therapeutic target in HCC.

Experimental study on siRNA expressing vector-based RNA interference targeting c-Myc in human hepatocellular carcinoma cell line BEL-7402

Objective: To explore the inhibition of the expression of c-Myc in human hepatocellular carcinoma via vector-based RNA interference (RNAi). Methods: Two RNA interference DNA templates targeting c-Myc oncogene were designed via online software and synthesized. By ligation, the fragments were inserted into pSilencer 1.0-U6 to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced into BEL-7402 cells with Lipofactamine. The inhibition of c-Myc expression, together with the expression of CDK4, hTERT and Gadd45β in c-Myc down-regulated BEL-7402 cells, were analyzed by semi-quantitative RT-PCR. Results: Two recombinant plasmids pSic-myc-1 and pSic-myc-2, which direct the yields of siRNAs targeting c-Myc in cells, were constructed. Among which, pSic-myc-2 was shown to trigger a RNAi-mediated inhibition of expression of c-Myc in BEL-7402 by up to 90%. In c-Myc knockdown BEL-7402 cells, the expression of CDK4 and hTERT were down-regulated with a ratio of 85% and 57%, respectively, while the expression of Gadd45β was up-regulated by up to 110%. Conclusion: The expression of c-Myc in BEL-7402 could be suppressed by vector-based RNA interference successfully. The knockdown of c-Myc in turn resulted in the changes of expression of genes related to cell proliferation and apoptosis. Thus, our study provided a preliminary data in searching of a c-Myc-targeted RNAi therapy of human hepatocellular carcinoma.

METTL5 stabilizes c-Myc by facilitating USP5 translation to reprogram glucose metabolism and promote hepatocellular carcinoma progression

Background:Hepatocellular carcinoma(HCC)is one of the most prevalent cancers in the world,with a high likelihood of metastasis and a dismal prognosis.The reprogramming of glucosemetabolism is critical in the development ofHCC.TheWarburg effect has recently been confirmed to occur in a variety of cancers,including HCC.However,little is known about the molecular biological mechanisms underlying the Warburg effect in HCC cells.In this study,we sought to better understand how methyltransferase 5,N6-adenosine(METTL5)controls the development of HCC and theWarburg effect.Methods:In the current study,quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of METTL5 in HCC tissues and cell lines.Several different cell models and animal models were established to determine the role of METTL5 in glucose metabolism reprogramming and the underlying molecularmechanism of HCC.Glutathione-S-transferase pulldown,coimmunoprecipitation,RNA sequencing,non-targeted metabolomics,polysome profiling,and luciferase reporter assays were performed to investigate the molecular mechanisms of METTL5 in HCC cells.Results:We discovered that METTL5 drove glucose metabolic reprogramming to promote the proliferation and metastasis of HCC.Mechanistically,upregulation of METTL5 promoted c-Myc stability and thus activated its downstream glycolytic genes lactate dehydrogenase A(LDHA),enolase 1(ENO1),triosephosphate isomerase 1(TPI1),solute carrier family 2 member 1(SLC2A1),and pyruvate kinase M2(PKM2).The c-Box and ubiquitin binding domain(UBA)regions of ubiquitin specific peptidase 5(USP5)binded to c-Myc protein and inhibited K48-linked polyubiquitination of c-Myc.Further study revealed that METTL5 controled the USP5 translation process,which in turn regulated the ubiquitination of c-Myc.Furthermore,we identified cAMP responsive element binding protein 1(CREB1)/P300 as a critical transcriptional regulator ofMETTL5 that promoted the transcription of METTL5 in HCC.In patient-derived tumor xenograft(PDX)models,adenovirus-mediated knockout of METTL5 had a good antitumor effect and prolonged the survival of PDX-bearing mice.Conclusions:These findings point to a novel mechanism by which CREB1/P300-METTL5-USP5-c-Myc controls abnormal glucose metabolism and promotes tumor growth,suggesting that METTL5 is a potential therapeutic target and prognostic biomarker for HCC.

Distribution of oval cells and c-myc mRNA expression in mouse hepatocarcinogenesis

BACKGROUND: This study was designed to assess theroles of oval cells and c-myc mRNA in the process of hepa-tocarcinogenesis and to clarify the function of carcinogenec-myc in the development of hepatocellular carcinoma( HCC) and the mechanism of inhibitory function of uscha-ridin on HCC in mouse hepatocarcinogenesis.METHODS: A total of 120 clean SD mice were divided intonormal group, cancer induction group, and interventiongroup. The normal group was fed with standard foragewhile the rest two groups were given p-dimethylaminoazo-benzene (DAB) to induce cancer. Thirteen weeks after in-duction of cancer, the two groups were fed with standardforage and water. Once the pattern was set up, the inter-vention group was given uscharidin injection into the ab-dominal cavity from the first week to the 14th week. Onthe 2nd, 4th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, 20th,22nd, and 24th week, all mice were killed and biopsiedfrom the liver lobe for pathological analysis. At the sametime, the number of tumor nodes was counted and the ex-pression of c-myc mRNA was tested by RT-PCR.RESULTS: Since the 2nd week after cancer induction, pro-liferated oval cells could be seen in the portal area. Initially,the oval cells appeared in the cortical layer of the portalarea, then proliferated gradually and immigrated into theliver parenchyma. In the period of fibrosis after liver proli-feration, proliferated heaps of oval cells were noted in bothportal and peripheral areas. In the period of carcinomatouschange, oval cells could be seen both outside and inside ofcancer nodes, but most of them were distributed outside.The c-myc gene was expressed negatively in the liver tissueof mice. The quantity of the expression began to increaseat the time of infection of the liver and tended to increasewith the degree of hepatic injury. In the period of cancera-tion, the expression level of c-myc mRNA increased gra-dually. The intervention of uscharidin could not inhibit butdelay the increase of the expression of c-myc mRNA.CONCLUSION: Oval cells are closely related to hepatocar-cinoma cells, which play an important role in the occur-rence and development of hepatocarcinogenesis. Uschari-din can inhibit the occurrence of hepatocarcinogenesis orlocal spreading at the early stage of cancer induction byDAB, but it cannot inhibit the expression of c-myc.